Cleavable linker for peptide synthesis

ABSTRACT

The present invention provides a new building block for peptide synthesis, which introduces a cleavage site that can be used to generate cleavable fragments subsequent to a peptide sequence.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. Pat. Application No.17/170,060 filed on Feb. 8, 2021, which application is a continuation ofInternational Application No. PCT/EP2019/071161 filed on Aug. 7, 2019,which application claims the benefit of the filing date of EuropeanPatent Application No. 18188135.0 filed on Aug. 9, 2018, the disclosuresof which are hereby incorporated by reference herein in theirentireties.

FIELD OF THE INVENTION

The present invention relates to the technical field of peptidesynthesis. More precisely, the present invention provides a newpossibility to introduce cleavable linkers into chemically synthesizedpeptides, thereby creating new peptide conjugates.

PRIOR ART

Cleavable linkers, defined as chemical moieties which connect twofunctionalities through a cleavable bondx, are important tools in solidphase synthesis (SPS) and chemical biology. Especially in solid phasepeptide synthesis (SPPS) these linkers can help solving issues regardingthe physicochemical properties, handling and purification of peptides:Through a cleavable linker peptides can be modified with functional tags(for example solubility enhancing moieties) and after the cleavage ofthe linker the desired peptide is released, with or without a residue ofthe linker. Cleavable linkers are widely used in organic synthesis andsolid phase synthesis (see for example Leriche et al., Bioorg. Med.Chem. 2012, 20, 571-582; Scott et al., Eur. J. Org. Chem. 2006,2251-2268). Cleavage may be performed by chemical (nucleophiles, basicreagents, electrophiles, acidic reagents, reducing reagents, oxidizingreagents, organometallic and metal catalysts), by photochemical orenzymatic means. In peptide synthesis cleavable linkers are mainly usedto link the nascent peptide to a resin which can be cleaved off aftercompletion of solid phase peptide synthesis (see for example NovabiochemPeptide Synthesis Catalogue, Merck; Jensen et al. (Ed.), PeptideSynthesis and Application, Methods in Molecular Biology, Vol. 1047,Springer Protocols, Humana Press, Springer, New York, 2013).

For internal incorporation into a peptide sequence cleavable linkerbuilding blocks have also been described. α,γ-Diamino-β-hydroxybutanoicacid and γ-amino-α,β-dihydroxybutanoic acid based linker building blocksfor peptide synthesis have been described by Amore et al., ChemBioChem2013, 14, 123-131. These linkers can be cleaved by oxidative means, i.e.using sodium periodate. Disadvantageous may be oxidation of oxidationsensitive components within the peptide like cysteine or methionineresidues.

Photocleavable linker building blocks for peptide synthesis which havebeen described by Kim et al., Synlett 2013, 24, 733-736 are anotherexample. Disadvantages of photoirradiation may be incomplete linkercleavage and side reactions arising from radical reactions. Acyclitively cleavable linker for alcohols based on[2-(aminomethyl)phenyl]acetic acid has been described by Xiao et al., J.Comb. Chem. 1999, 1, 379-382. This linker has only been applied for thesynthesis and release of alcohols by using a solid support. A derivativewhich can be used for internal incorporation in peptide synthesis hasnot been described. Peptide synthesis applying cleavable solubilizingtags has been described using different chemistries and cleavageconditions (see for example: Jacobsen et al., JACS 2016, 138,11777-11782; WO 2016047794).

Peptide synthesis applying cleavable purification tags has beendescribed using different chemistries and cleavage conditions (see forexample: Funakoshi et al., Proceedings of the National Academy ofSciences 1991, 88, 6981-6985; Funakoshi et al., J. Chromatogr. 1993,638, 21-27; Canne, et al. Tetrahedron Letters 1997, 38, 3361-3364;Vizzavona et al., Tetrahedron Letters 2002, 43, 8693-8696; Hara et al.,Journal of Peptide Science, 2009, 15, 369-376; Aucagne et al.,Angewandte Chemie International Edition 2012, 51, 11320-11324; Reimannet al., Angewandte Chemie International Edition 2015, 54, 306-310; Haraet al., Journal of Peptide Science, 2016, 15, 379-382; Patents: Aucagneet al. WO 2011058188, Zitterbart et al. WO 2017129818 A1). In thisapproach, the linkers are usually attached in the last cycle of SPPS tothe N-terminus of the growing peptide chain to enable selectiveimmobilization of the desired full-length peptide onto a solid support.Side products are removed by washing the solid support and eventuallythe target peptide is released by cleavage of the linker. The cleavablelinkers used for non-chromatographic purification of peptides usuallyrequire strong basic conditions. Under these conditions undesiredside-reactions might occur e.g. racemization. A major problem of thefrequently used sulfonate-elimination linkers are the highly reactiveelectrophiles, which are generated during the cleavage reaction. Theseintermediates react rapidly with the nucleophilic side-chain groups(e.g. arginine, cysteine) and consequently limit the application of thesulfonate based cleavable linkers. The rather rare example of anoxidatively cleavable purification linker from Vizzavona et al.(Tetrahedron Letters 2002, 43, 8693-8696) circumvent the aforementionedproblems, but is most likely not compatible with methionine or cysteinecontaining peptides.

Isoacyl dipeptides are tools for enhancing synthetic efficiency in FmocSPPS (Y. Sohma et al., Chem. Commun. 2004, 124-125). Isoacyl dipeptidesconsist of a Boc-protected serine or threonine derivative in which theβ-hydroxyl group is acylated by a Fmoc-protected amino acid. Afterincorporation of an isoacyl dipeptide building block within the sequenceof a peptide, the secondary structure of the peptide is changed enablingmore efficient synthesis. Furthermore, after cleavage and deprotectionthe isoacyl form of the peptide can be purified by HPLC. At pH 7.4 O→Nintramolecular acyl migration takes place to generate the regularlyamide linked peptide. Applying these isoacyl dipeptide building blocksno cleavage reaction can be performed.

Peptide-oligonucleotide conjugates are an emerging class for therapeuticand diagnostic applications. However, the synthesis of these conjugatesremains a major challenge (see reviews of N. Venkatesan et al., ChemicalReviews 2006, 106, 3712-3761 and K. Lu et al., Bioconjugate Chemistry2010, 21, 187-202). A straight forward approach would be to assemble thedesired peptide-oligonucleotide conjugates on a polymeric support bymeans of solid-phase based synthesis. Unfortunately, established methodsof solid-phase oligonucleotide and peptide synthesis are not fullycompatible. The solid-phase synthesis of peptides requires the use ofstrong acids and thereby prevents the synthesis ofpeptide-oligonucleotide conjugates due to instability ofoligonucleotides under acidic conditions. This is why the stepwisesynthesis of peptide-oligonucleotide conjugates usually proceeds byfirst assembling the peptide, followed by oligonucleotide synthesis onthe same solid support. Albeit this strategy has been successfullyapplied for the synthesis of rather simple peptide-oligonucleotideconjugates, the method is still lacking the full spectrum of compatibleprotecting groups to address the challenging chemistry of the aminoacids side chains. Conclusively, a reliable and general applicablemethod for the stepwise solid-phase based synthesis ofpeptide-oligonucleotide conjugates is not available. Therefore, thesynthesis of peptide-oligonucleotide conjugates usually proceeds byemploying a convergent strategy. Here the peptide and theoligonucleotide fragments are synthesized separately by using routinebuilding blocks and protocols of solid-phase synthesis. Afterpurification the two fragments are conjugated and the desiredpeptide-oligonucleotide conjugate is isolated after an additionalpurification step. Low overall yields, increased expenditure of time andhigh costs are the major disadvantages of this strategy, which resultfrom the aforementioned numerous purification steps and intermediatelyophilization procedures. Moreover, HPLC-based purification steps andintermediate lyophilization imped the possibility of achieving ahigh-throughput synthesis of peptide-oligonucleotide conjugates by meansof automation. These drawbacks become particularly troublesome, if alarge number of peptide-oligonucleotide conjugates needs to besynthesized, which is required e.g. for screening a suitabletransfection peptide on a known antisense oligonucleotide. A perfectmethod would combine the ease of established solid-phase synthesis andpost-synthetic conjugation while bypassing the need of any HPLC-basedpurification.

However, all the above disclosed methods for applying cleavable linkersfor peptide synthesis have several disadvantages like inefficientincorporation or cleavage, harsh, damaging cleavage conditions, complexreagent synthesis or restricted use.

BRIEF DESCRIPTION OF THE INVENTION

The present invention therefore provides a building block comprising thestructure

wherein 2 ≤ n ≤ 24, m = 2 or 3, and A and B are protective groups.Usually, A and B are orthogonal protective groups which are cleavedunder different conditions. In one embodiment, A is an acid labileprotective group and

B is a tag or base labile protective group. In one particularembodiment, A is Boc and/or B is Fmoc.

In a second aspect, the present invention provides a compound comprisingthe structure

wherein 2 ≤ n ≤ 24, m = 2 or 3, X is a peptide, or a solid support and Yis selected from a group consisting of a peptide, a functional group, atag, and a peptide containing a functional group or a tag.

In one embodiment, Y is either a solubility enhancing tag, animmobilization tag or a solid phase. For example, Y may be selected froma group consisting of PEG, poly-lysine, poly-arginine, poly-glutamicacid, and poly-aspartic acid. Y may also be selected from a groupconsisting of biotin, hydrazine, aminooxy, azide, alkynyl, alkenyl,aldehyde, ketone, pyrroloalanine, carboxy and thiol.

In a third aspect, the present invention provides a method comprisingthe steps of

-   synthesizing a peptide on a solid support, said peptide comprising a    terminal amino group,-   providing a building block as disclosed above, and-   coupling said building block to said peptide.-   Said method may further comprise the steps-   removing protective group B, and-   coupling at least one amino acid building block to the terminal    amino group.-   Alternatively, said method may further comprise steps removing    protective group B,-   optionally coupling at least one amino acid building block to the    terminal amino group, and coupling a tag or a functional group to    the terminal amino group.-   Said functional group or tag may be selected from a group consisting    of PEG, poly-lysine, poly-arginine, poly-glutamic acid,    poly-aspartic acid, biotin, hydrazine, aminooxy, azide, alkynyl,    alkenyl, aldehyde, pyrroloalanine, carboxy, and thiol.

In addition, the methods disclosed above may further comprise the stepof

removing protective group A at a pH ≤ 6, thereby also removing otherprotective groups present on said peptide and cleaving said peptide fromthe solid support, which may occur at a pH ≥ 8.

In one embodiment, the present invention provides a method comprisingthe steps of

-   a) synthesizing a peptide on a solid support, said peptide    comprising a terminal amino group,-   b) providing a building block as disclosed above, and-   c) coupling said building block to said peptide-   d) removing protective group B,-   e) optionally coupling at least one amino acid building block to the    terminal amino group, and-   f) coupling a solubilizing or immobilizing tag to the terminal amino    group,-   and further comprising the steps-   g) removing protective group A at a pH ≤ 6, thereby also removing    other protective groups present on said peptide and cleaving said    peptide from the solid support-   h) purifying said peptide, and-   i) cleaving off said solubilizing tag at a pH ≥ 8, or-   g) removing protective group A at a pH ≤ 6, thereby also removing    other protective groups present on said peptide and cleaving said    peptide from the solid support-   h) immobilizing said peptide via said immobilizing tag on a solid    support-   i) optionally conjugating said peptide to an additional chemical    entity, and-   j) cleaving off said immobilizing tag at a pH ≥ 8.

Said chemical entity may be a carbohydrate, a protein, a peptide, a dye,a hapten, or the like. In particular, said chemical entity may be anucleic acid, oligonucleotide or nucleotide containing compound,preferably a nucleoside-hexaphosphate.

BRIEF DESCRIPTION OF FIGURES

FIGS. 1A - 1C illustrate peptide synthesis according to example 3, wherethe method comprises introduction of a linker.

FIG. 1A illustrates peptide synthesis.

FIG. 1B illustrates solvation in 0.05 M NaHCOs solution (pH = 8.2),triggering cyclization.

FIG. 1C depicts a series of LC-MS chromatograms showing cleavage ofpeptide AB into A and B over time.

FIGS. 2A - 2C illustrate the synthesis of a hydrophobic peptideaccording to example 4 (SEQ ID NO: 3).

FIG. 2A depicts the cleavage of poly-lysine tag from insoluble peptide.

FIG. 2B provides a LC-MS chromatogram of H-KKKKK1AhaGISFSIRFAIWIRFG-NH2(10) (SEQ ID NO: 3).

FIG. 2C provides a MS (ESI) of insoluble peptide 12.

FIGS. 3A - 3C depict affinity purification according to example 5 (SEQID NO: 4).

FIG. 3A depicts a crude peptide mixture after SPPS and cleavage fromresin.

FIG. 3B illustrates supernatant after 30 min.

FIG. 3C depicts supernatant after incubation with 0.02 M NH₄HCO₃(pH=8.8) for 30 min.

FIG. 4 sets forth a synthetic scheme, namely the rapid synthesis ofnucleoside-peptide conjugates according to example 7. In particular, theFIG. 4 depicts the synthesis of peptides and nucleoside-peptideconjugates by solid-supported conjugation and non-chromatographicpurification.

FIGS. 5A - 5D set forth HPLC-analyses of conjugates obtained in example7.

FIG. 5A depicts a chromatogram of the crude material after SPPS.

FIG. 5B depicts a chromatogram after washing the supernatant containingdeletions.

FIG. 5C depicts a chromatogram of the pure peptide.

FIG. 5D depicts a chromatogram of the pure peptide nucleotide conjugate.

SEQUENCE LISTING

The contents of the electronic sequence listing (Ventana-0211US1.xml;Size: 8,456 bytes; and Date of Creation: Nov. 2, 2022) is hereinincorporated by reference in its entirety.

DETAILED DESCRIPTION OF THE INVENTION

List of definitions and abbreviations:

-   Fmoc: Fluorenylmethyloxycarbonyl-   Boc: tert-Butyloxycarbonyl-   SPPS: Solid Phase Peptide Synthesis-   SPS: Solid Phase Synthesis-   TFA: Trifluoroacetic acid-   DBU: 1,8-Diazabicyclo[5.4.0]undec-7-ene-   DMF: N, N-Dimethylformamide-   DAB: Diamino butyric acid-   EDC: N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide-   DMAP: N,N-Dimethylpyridin-4-amine-   HATU:    1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium    3-oxid hexafluorophosphate-   DIPEA: N,N-Diisopropylethyl amine-   THPTA: Tris(3-hydroxypropyltriazolylmethyl)amine-   Pra: Propargylglycine-   Aha: Azidohomoalanine-   TIS: Triisopropyl silane

Definitions:

-   Tag: In the context of the present invention, a tag is a chemical    moiety which alters the chemical or physical properties of a    molecule and/or renders the molecule recognizable. For example, a    purification tag may facilitate purification of a molecule. A tag    may be an immobilization tag, i.e. a chemical moiety that can be    attached to a solid support. A tag may also be a solubility    enhancing tag, that means a chemical group which if present    increases the solubility of a certain molecule.-   Functional Group: Functional groups are specific chemical groups    (moieties) of atoms or bonds within molecules that are responsible    for the characteristic chemical reactions of those molecules.    Functional groups are specific substituents or moieties within    molecules that are responsible for the characteristic chemical    reactions of those molecules. The same functional group will undergo    the same or similar chemical reaction(s) regardless of the size of    the molecule it is a part of. This allows for systematic prediction    of chemical reactions and behavior of chemical compounds and design    of chemical syntheses. Furthermore, the reactivity of a functional    group can be modified by other functional groups nearby. In organic    synthesis, functional group interconversion is one of the basic    types of transformations. Functional groups are groups of one or    more atoms of distinctive chemical properties no matter what they    are attached to. The atoms of functional groups are linked to each    other and to the rest of the molecule by covalent bonds. For    repeating units of polymers, functional groups attach to their    nonpolar core of carbon atoms and thus add chemical character to    carbon chains. Functional groups can also be charged.-   Protecting group: The term “protective group” or its synonym    “protecting group” denotes the group which selectively blocks a    reactive site in a multifunctional compound such that a chemical    reaction can be carried out selectively at another unprotected    reactive site in the meaning conventionally associated with it in    synthetic chemistry. Protecting groups can be removed at the    appropriate point. Protecting groups are amino-protecting groups,    carboxy-protecting groups or hydroxy-protecting groups. A protecting    group or protective group or blocking group is introduced into a    molecule by chemical modification of a functional group to obtain    chemoselectivity in a subsequent chemical reaction. A protecting    group is introduced to block or at least reduce the reactivity of    functional groups. A deprotection is a chemical step of removal of a    protecting group. Relevant protective groups in the field of peptide    synthesis are base labile protecting groups and acid labile    protecting groups. Base labile protecting groups are cleaved off at    a pH between 7.5 and 12, but preferably at a pH between 8.0 and 10.    Acid labile protective groups are cleaved off at a pH between 6.5    and 3, but preferably at a pH between 6.0 and 5. For the present    invention, amino protecting groups are of particular importance.    Amino-protecting groups are groups intended to protect an amino    group and includes benzyl, benzyloxycarbonyl (carbobenzyloxy, CBZ),    Fmoc (9-Fluorenylmethyloxycarbonyl), p-methoxybenzyloxycarbonyl,    p-nitrobenzyloxycarbonyl, tert-butoxycarbonyl (BOC), and    trifluoroacetyl. Examples of these groups are found in T. W. Greene    and P. G. M. Wuts, “Protective Groups in Organic Synthesis,” 2nd    ed., John Wiley & Sons, Inc., New York, N.Y., 1991, chapter 5; E.    Haslam, “Protective Groups in Organic Chemistry”, J. G. W. McOmie,    Ed., Plenum Press, New York, N.Y., 1973, Chapter 5, and T.W. Greene,    “Protective Groups in Organic Synthesis”, John Wiley and Sons, New    York, NY, 1981, Chapter 5.-   Peptide: A peptide is a chain of amino acid building blocks linked    through amide bonds with a length of 2 to 120 residues.

Novel cleavable linkers for peptide synthesis have been developed.Cleavage occurs under mild basic conditions. The linkers are based on a4-aminobutanoate core which undergoes intramolecular lactamization atpH >8 cleaving the ester bond by releasing two fragments, the N-terminalalcohol and the C-terminal lactam (Scheme 1A). As Nα-Fmoc-Nγ-Boc-protected building block (Scheme 1B) this aminobutanoatecleavable linker can be employed in solid phase peptide synthesis.Aminobutanoate linker 1 turned out to be stable during conventionalpeptide synthesis and surprisingly during an Fmoc deprotection of thesuccessive amino acid no cyclization to a 6 membered diketopiperazineand the corresponding breakup peptide was observed.

Schemes 1A and 1B

-   a) Cyclitively cleavage of the linker (n> 1).-   b) General formula of protected cleavable linker (n > 1).

During solid phase peptide synthesis the Nγ-Boc protected amino group isunreactive and aminobutanoate linker remains intact. Cleavage of thepeptide from the solid support and deprotection of protecting groupsunder acidic conditions leads to removal of the Ny-Boc protecting groupof the aminobutanoate linker. Since the amino group is protonated underthe acidic cleavage and deprotection conditions the lactamizationreaction is fully suppressed and the aminobutanoate linker remainsintact. The peptide containing the intact aminobutanoate linker cantherefore also be purified under acidic conditions (i.e. water/acetonitrile/ trifluoroacetic acid eluent). Under mild basic conditionsthe cleavage of the aminobutanoate linker proceeds by means of anintramolecular cyclization reaction, releasing two peptides, one as aN-alcohol and the other as a C-terminal lactam. The general concept isshown in Scheme 2 which shows solid phase peptide synthesis with anaminobutanoate linker (n> 1) and final cleavage with release of twopeptide fragments.

Scheme 2

It has been found that a cleavable linker with n = 1 is not suited sinceside reactions occurred during solid phase peptide synthesis (i.e. estercleavage). However, cleavable linkers with n = 2 and n = 3 werecompatible with solid phase peptide synthesis and yielded the desiredlinker-containing peptides, which could be cleaved under mild alkalineconditions. Therefore the above mentioned building blocks can be used toadd a variety of functional groups to the N-terminus of a peptide whichcan be removed at a later process step.

One application of adding a cleavable functional group is to introduce ahydrophilic tag onto the N-terminus of a hydrophobic peptide in order toenable the purification (i.e. by HPLC) of such a hydrophobic peptide.After purification, the solubilizing tag can be cleaved off under mildbasic conditions in order to release the purified hydrophobic targetpeptide.

The method can also be applied for an improved synthesis ofoligonucleotide-peptide conjugates. This is of particular interest ifthe peptide contains many hydrophobic residues. For instance, thehydrophobic peptide can be synthesized first, containing a conjugationsite for the attachment of the oligonucleotide, such asazidohomoalanine. Then the cleavable aminobutanoate linker is coupled,followed by introduction of a solubilizing tag sequence. After cleavageand deprotection the peptide can be purified by HPLC and finally beconjugated to an oligonucleotide functionalized with a conjugation site.Said conjugation site, for example, may be an alkyne group. Thereafterthe conjugate can be purified and the solubilizing tag can be cleavedoff under mild alkaline conditions.

Another application is the introduction of a purification tag which maybe introduced at the N-terminus of a peptide. Biotin is a prominentexample for such a purification tag. First, peptide is synthesized viaSPPS including a capping step after each coupling. Thereafter thecleavable linker is coupled to the N-terminus of the peptide and finallybiotin is coupled onto the cleavable linker. After cleavage anddeprotection under acidic conditions, the biotin labeled peptide can bebound to streptavidin coated beads, which are preferably magnetic beads.Non-biotinylated by-products of SPPS (i.e. failure sequences, deletions)can be removed by washing. Thereafter, the purified peptide can bereleased from the streptavidin beads by cleaving the linker under mildalkaline treatment.

Contemporary methods for the convergent synthesis ofpeptide-oligonucleotide conjugates require multiple purification steps.Therefore, the screening of numerous peptide-oligonucleotide conjugatesis a time-and cost-consuming endeavor. The method of the inventionenables the rapid synthesis of peptide-(oligo)nucleotide conjugates. Themethod of the invention using the cleavable linker building block of theinvention circumvents the need of tedious HPLC-purification steps by thecombination of chemoselective reactivity units and cleavablepurification tags and allowing for mild cleavage of the molecule ofinterest. This non-chromatographic purification approach enables theparallel synthesis of numerous conjugates in good yield and purity.

As will be shown within the examples, the cleavable linker of theinvention is easy to synthesize, allows efficient incorporation intopeptides and mild cleavage under slightly alkaline, non-destructingconditions.

Examples

Example 1: Synthesis of linker 1 (m=2; n=3)

Scheme 3 Synthesis of Compound 2:

Allyl 4-hydroxybutanoate

To a solution of γ-butyrolactone (5.00 g, 58.1 mmol) in DMF (17 ml) wereadded H₂O (13.6 g, 13.6 ml, 755 mmol) and DBU (8.85 g, 8.67 ml, 58.1mmol). After 1 h stirring at r.t. allyl bromide (10.5 g, 7.53 ml, 87.2mmol) was added to the solution. The reaction was quenched after 1 h byaddition of sat. aq. NH₄Cl solution (30 ml) and the aqueous phase wasextracted with ethyl acetate (3 x 100 ml). The combined organic layerswere dried over Na₂SO₄ and the solvent was removed under reducedpressure. Column chromatography over SiO₂ (n-hexane/ethyl acetate = 1:1)afforded the desired product (5.90 g, 40.9 mmol, 71%) as a colorlessoil.

Rf (n-hexane/ethyl acetate = 1:1) = 0.33 (KMnO₄)

¹H-NMR (400 MHz, CDC1₃): δ = 6.06 - 5.86 (m, 1H), 5.42 - 5.04 (m, 2H),4.59 (td, J = 1.4, 5.7 Hz, 1H), 4.35 (t, J = 7.0 Hz, 1H), 4.25 - 4.06(m, 1H), 3.73 - 3.67 (m, 1H), 2.55 - 2.41 (m, 2H), 2.34 - 2.16 (m, 1H),1.99 - 1.83 (m, 1H), 1.62 (s, 1H).

Synthesis of Compound 3

4-(Allyloxy)-4-oxobutyl2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-((tert-butoxycarbonyl)amino)butanoate

Commercially available Fmoc-Dab(Boc)-OH (1.00 g, 2.27 mmol) and compound2 (360 mg, 2.50 mmol) were dissolved in CH₂C1₂ (7.5 ml) and cooled to 0°C. EDC-HCl (479 mg, 2.50 mmol) and DMAP (28 mg, 0.227 mmol) were addedto the solution. After 1 h stirring at r.t. sat. aq. NaCl solution (25ml) was added to the reaction mixture which was then extracted withCH₂CI₂ (3 x 50 ml). The combined organic layers were dried over Na₂SO₄and the solvent was removed under reduced pressure affording the desiredproduct (1.27 g, 2.24 mmol, 99%) as a colorless resin.

Rf (n-hexane/ethyl acetate = 1:1) = 0.63

¹H-NMR (400 MHz, CDC13): δ = 7.80 - 7.72 (m, 2H), 7.64 - 7.56 (m, 2H),7.44 - 7.36 (m, 2H), 7.35 - 7.28 (m, 2H), 5.95 - 5.84 (m, 1H), 5.68 -5.57 (m, 1H), 5.34 - 5.20 (m, 2H), 5.10 - 4.98 (m, 1H), 4.61 - 4.55 (m,2H), 4.46 - 4.32 (m, 3H), 4.26 - 4.15 (m, 3H), 3.47 - 3.30 (m, 1H),3.02 - 2.93 (m, J=5.3, 5.3, 8.3, 14.0 Hz, 1H), 2.46 - 2.37 (m, 2H),2.12 - 1.94 (m, 3H), 1.82 - 1.72 (m, 1H), 1.51 - 1.35 (m, 9H).

MS (ESI): found 567.3 [M + H]⁺, 467.3 [M + H - Boc]⁺, calculated 567.3[M + H]⁺

Synthesis of Linker 1:

4-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-((tert-butoxycarbonyl)amino)butanoyl)oxy)butanoicacid

To a solution of 4-(allyloxy)-4-oxobutyl2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-((tert-butoxycarbonyl)amino)butanoate3 (4.5 g, 7.94 mmol) in CH₂C1₂/ethyl acetate 2:1 (75 ml) were addedtetrakis(triphenylphosphine)palladium(0) (275 mg, 0.238 mmol),triphenylphosphine (104 mg, 0.395 mmol) and sodium 2-ethylhexanoate(1.97 g, 11.9 mmol). The reaction was stirred at r.t. for 3 hours. Then1 M HCl (50 ml) was added. The reaction mixture was extracted withCH₂CI₂ (3 x 150 ml). The combined organic layers were dried over Na₂SO₄and the solvent was removed under reduced pressure. Columnchromatography over SiO₂ (ethyl acetate + 1 % methanol) afforded thedesired product 1 as a colorless solid.

Rf (ethyl acetate) = 0.38

¹H-NMR (400 MHz, CDC1₃): δ = 7.79 - 7.73 (m, 2H), 7.64 - 7.55 (m, 2H),7.44 - 7.37 (m, 2H), 7.35 - 7.28 (m, 2H), 5.70 - 5.54 (m, 1H), 5.18 -5.02 (m, 1H), 4.47 - 4.33 (m, 3H), 4.28 - 4.16 (m, 3H), 3.43 - 3.32 (m,1H), 3.04 - 2.94 (m, 1H), 2.55 - 2.25 (m, 3H), 2.15 - 1.98 (m, 3H),1.48 - 1.38 (m, 9H).

MS (ESI): found 527.3 [M + H]⁺, 427.2 [M + H - Boc]⁺, calculated 527.2[M + H]⁺

Example 2: Synthesis of linker 4 (m=2; n=2)

Synthesis of Compound 5:

3-(benzyloxy)-3-oxopropyl2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-((tert-butoxycarbonyl)amino)butanoate

Commercially available Fmoc-Dab(Boc)-OH (2.5 g, 5.68 mmol) and benzyl3-hydroxypropanoate (1.12 g, 6.25 mmol) were dissolved in CH₂Cl₂ (20 ml)and cooled to 0° C. To this solution EDC-HCl (1.20 g, 6.25 mmol) andDMAP (70.0 mg, 0.568 mmol) were added. After 1 h sat. aq. NaCl solution(25 ml) was added, the organic phase was separated and the aqueous phasewas extracted with CH₂CI₂ (3 x 50 ml). The combined organic layers weredried over Na₂SO₄ and the solvent was removed under reduced pressure.Column chromatography over SiO₂ (n-hexane/EtOAc 6:4) afforded thedesired product (2.84 g, 4.71 mmol, 83 %) as a colorless resin.

¹H-NMR (400 MHz, CDC1₃) δ = 7.78 - 7.74 (m, 2H), 7.65 - 7.58 (m, 2H),7.35 (s, 9H), 5.77 - 5.66 (m, 1H), 5.17 - 5.14 (m, 2H), 5.13 - 5.04 (m,1H), 4.54 - 4.46 (m, 1H), 4.45 - 4.30 (m, 4H), 4.26 -4.20 (m, 1H),3.91 - 3.87 (m, 1H), 3.46 - 3.33 (m, 1H), 2.97 - 2.88 (m, 1H), 2.77 -2.70 (m, 2H), 2.05 - 2.03 (m, 1H), 2.01 - 1.92 (m, 1H), 1.79 - 1.70 (m,1H), 1.47 - 1.42 (m, 9H).

Synthesis of Compound 4

3-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-((tert-butoxycarbonyl)amino)butanoyl)oxy)propanoicacid

Compound 4 (2.8 g, 4.65 mmol) was dissolved in MeOH (50 ml). Palladiumon carbon (Pd/C 10%, 742 mg, 0.69 mmol) was added to the solution and H₂was bubbled into the reaction mixture. After 1 h the reaction wasdiluted with CH₂CI₂ and filtered through a silica plug. Solvent wasremoved under reduced pressure affording the desired product (0.6 g,1.17 mmol, 25%) as a colorless solid.

MS (ESI): 513.0 [M + H] , calculated 513.2 [M + H]⁺.

Example 3 Peptide Synthesis Applying Linker 1

A peptide with the sequence H-KATSG - (linker1) - GLF-NH₂ (7) (SEQ. ID.No: 1) was synthesized by solid phase peptide synthesis and purified bypreparative HPLC. Linker 1 was stable during peptide synthesis, andsurprisingly also during Fmoc deprotection with piperidine of thesuccessive amino acid (Gly at position 5). No cyclization to a 6membered diketopiperazine and the corresponding breakup peptide wasobserved (FIG. 1A). The purified peptide (AB in FIG. 1B) was thendissolved in 0.05 M NaHCOs solution (pH = 8.2) which triggered theintramolecular cyclization reaction releasing the two fragments 8 and 9(A and B, FIG. 1B), identified by LC-MS (FIG. 1C).

Example 4 Synthesis of Hydrophobic Peptide

H-AhaGISFSIRFAIWIRFG-NH₂ (Aha = azidohomoalanine) (SEQ. ID. NO: 2) is anextremely hydrophobic peptide, which is insoluble in water, making thehandling and HPLC purification after synthesis virtually impossible. Inorder to enhance solubility of this peptide a variant was prepared inwhich a cleavable N-terminal poly-lysine tag was synthesized after thepeptide sequence and incorporated cleavable linker 1: H-KKKKK- (linker1)AhaGISFSIRFAIWIRFG-NH₂ (10) (SEQ. ID. No: 3). Synthesis andpurification of this modified peptide proceeded smoothly according toexample 3 (FIG. 2B). Pure peptide 10 was dissolved in a 0.05 M aqueousNaHCO₃ solution and shaken for 2 h at r.t. (FIG. 2A). The cleavagereaction released the two peptides 11 and 12: While the poly-lysine tag11 is water soluble, peptide 12 precipitated under the reactionconditions and could be easily isolated in high purity bycentrifugation.

FIG. 2B: LC-MS of H-KKKKK (linker 1)-AhaGISFSIRFAIWIRFG-NH₂ (10), MS(ESI): 545.4 [M+5H]⁵ ⁺, 681.6 [M+4H] ⁴⁺, 908.3 [M+3H]³⁺, 1362.0[M+2H]²⁺.

FIG. 2C: MS (ESI) of insoluble peptide 12 (MS (ESI): 661.5 [M+3H] ³⁺,991.3 [M+2H]²⁺, 1322.0 [3M + 2H]²⁺, 1983.1[M+H]⁺).

Example 5: Affinity Purification via Biotin / Streptavidin Interaction

The introduction of N-terminal affinity labels in combination with apeptide synthesis method applying capping after each coupling stepenables affinity purification of full-length products. The full-lengthpeptide is captured on streptavidin coated magnetic beads, by-products(shorter sequences) are removed by filtration and the pure full-lengthpeptide is released.

After SPPS of the target peptide, first linker 1, and thenFmoc-Glu(biotinyl-PEG)-OH were coupled to the N-terminus of the peptide.This resulted in H-GluBiotinylPEG-1-IIKKSTALL-NH₂ (13) (SEQ. ID. NO: 4).As the peptide sequence contains several sterically demanding aminoacids, a complex mixture of full-length peptide and acetylated shorterfragments was obtained after cleavage from the resin. The crude productwas dissolved in a phosphate buffer at pH = 6.2 and incubated for 30minutes with streptavidin coated magnetic beads. The supernatant wasanalyzed by LC-MS indicating the complete removal of the biotinylatedpeptide from the mixture. The byproduct containing buffer solution wasremoved and the beads were washed several times with phosphate buffer(pH = 6.2). Afterwards a volatile cleavage buffer (NH₄HCO₃, 0.02 _(M),pH = 8.8) was added to the beads and after 30 min incubation the desiredpeptide 14 was released from the beads.

Results are shown in FIG. 3 . FIG. 3A shows the crude peptide mixtureobtained after SPPS and cleavage from resin. FIG. 3B shows thesupernatant after 30 min incubation of the crude peptide mixture onstreptavidin coated magnetic beads in phosphate buffer at pH = 6.2. TheN-terminally biotin tag containing peptide 13 is completely captured bystreptavidin. c) After washing away the acetylated peptide by-productsand incubating the magnetic streptavidin beads with NH₄HCO₃ at pH 8.8for 30 min to release the desired peptide HO(CH₂)₃CO—IIKKSTALL—NH₂ (14)(SEQ. ID. NO: 4)

Example 6: Synthesis of DNA-peptide conjugates employing cleavablesolubilizing tag on peptide

A peptide with the sequence H-PEG10 (linker 1) Aha AFDYLAQYHGG-NH₂ (15)(SEQ ID No: 5) was synthesized by SPPS (Aha = azidohomoalanine).Conjugation with the hexynyl-modified nucleic acid was performed byclick chemistry. A solution of the azido-modified peptide 15 (4 mM inDMSO/tBuOH 3:1, 50 µl) was mixed with a solution of 5'-hexynyl-dT₂₀-3'(0.55 mM in H₂O, 180 µl) which was synthesized according to standardsolid phase phosphoramidite approach. CuBr (100 mM in DMSO/tBuOH 3:1, 10µl) and THPTA (100 mM in H₂O, 20 µl) were mixed separately and thepreformed complex was added to the oligonucleotide-peptide solution.After 1 h shaking at 37° C. the click reaction was complete. Cleavage ofthe solubilizing PEG linker was obtained adding NaHCO₃ (0.1 _(M) in H₂O,3 ml). Dialysis of this solution (MWCO 1000 dialysis membrane) affordedthe desired product 16.

5'-hex-T₂₀-3': MS (ESI): found 1544.3 [M - 4H]⁴⁻, calculated 1544.5 [M -4H]⁴⁻.

Peptide X - 5'- hex-T₂₀-3': MS (ESI): found 1647.7 [M - 5H]⁵⁻,calculated 1648.0 [M - 5H]⁵⁻.

Product 16: Peptide X - 5'- hex-T₂o-3'(Depegilated): MS (ESI): found1525.6 [M - 5H]⁻⁵, calculated 1525.9 [M - SH]⁻⁵.

Example 7: Rapid Synthesis of Peptides and Nucleoside-peptide Conjugatesby Solid-Supported Conjugation and Non-Chromatographic PurificationUsing a Cleavable Immobilization Linker

The reaction scheme disclosed in this example is illustrated in FIG. 4and comprises two alternative routes A and B.

The peptide WWWWEAAAEAAAEAAAEAAAEAAAEAAAEAAAEAAAE-AAAEAAAEAAAEAAAEEEEE(SEQ. ID. No: 6) was synthesized on a rink-amide resin using automatedFmoc-SPPS. Next, Fmoc-Pra-OH(N-alpha-(9-Fluorenylmethyloxycarbonyl)-L-propargylglycine),Fmoc-protected cleavable linker 1, Fmoc-O2Oc-OH(8-(9-Fluorenylmethyloxycarbonyl-amino)-3,6-dioxaoctanoic acid) andTri-Boc-hydrazine acetic acid were stepwise coupled to the resin-boundpeptide by means of SPPS, yielding the desired peptide 17.

The peptide was cleaved from the resin with a mixture of TFA/TIS/H₂O(95/2.5/2.5). After 2 h the solution was concentrated and added to asolution of cold ether. The precipitated peptide was separated from thesupernatant, dissolved in a mixture of H₂O/ACN (1/1) and freeze-dried.The crude material was dissolved in a mixture of an aqueous 0.25 MNaOAc/AcOH buffer (pH 4.2) and acetonitrile (20 vol.%). Sodiumcyanoborohydride was added and the solution was transferred onto analdehyde agarose resin. The suspension was agitated at room temperatureand the progress of the immobilization reaction was monitored by HPLC(see FIGS. 5A and 5B). After immobilization (~1 h), the resin was washedwith an aqueous 0.25 M NaOAc/AcOH buffer (pH 4.2), a mixture of H₂O/ACN(2/1) and water. The resin-bound propargylglycine-containing peptide 18was brought into reaction with the azide-modified hexaphosphatethymidine (Fuller et al. PNAS 2016, 113 (19), p.5233-4238) in presenceof CuSO₄, THPTA, ascorbate and aminoguanidine in a mixture of an aqueous0.2 M NaH₂PO₄ buffer (pH 6.5) containing 20 vol.% DMSO (see FIG. 4 ,route A). The suspension was agitated at 37° C. for 16 h. The resin waswashed with an aqueous 0.2 M NaHPO₄ buffer (pH 6.5) and a mixture ofH₂O/ACN (2/1). The desired Nucleoside-peptide conjugate 20 was releasedin good purity by treatment of the resin with an aqueous 0.2 M Na₂HPO₄buffer (pH 8.5) for 16 h at room temperature.

(HO(CH₂)₃CONH-Pra(N₃(CH₂)₁₁O(PO₂)₆dT)

WWWWEAAAEAAAEAAAEAAAEAAAEAAAEAAAEAAAEAAA-EAAAEAAAEAAAEEEEE -NH₂:

MS (ESI): found 1101.3 [M - 6H]⁶⁻, calculated 1101.2 [M - 6H]⁶⁻ ; found944.0 [M - 7H]⁷⁻, calculated 943.8 [M - 7H]⁷⁻.

Alternatively, the resin bound peptide 18 could be also released undermild basic conditions (0.2 M Na₂HPO₄ buffer, pH 8.5, r.t., 16 h) fromthe agarose resin (see FIG. 4 , route B) even before thenucleoside-conjugation step. Under these conditions, the unmodifiedpeptide 19

HO(CH₂)₃CONH-PraWWWWEAAAEAAAEAAAEAAAEAAAEAAAEAAAEAAAEAAA-EAAAEAAAEAAAEEEEE -NH₂ wasisolated in high purity.

Product generation was monitored by HPLC analysis. FIG. 5 showsHPLC-analysis of the crude SPPS product (5A), the supernatant afterimmobilization (5B), the purified peptide 19 (5D) generated according toroute B and the nucleoside-peptide conjugate 20 (5C).

Additional Embodiments

Additional Embodiment 1. A building block comprising the structure

wherein 2 < n < 24, m = 2 or 3,

A is an acid labile protective group and

B is a tag or base labile protective group.

Additional Embodiment 2. A building block according to additionalembodiment 1, wherein A is Boc and/or B is Fmoc.

Additional Embodiment 3. A compound comprising the structure

wherein

wherein 2 ≤ n ≤ 24, m = 2 or 3,

X is a peptide, or a solid support and

Y is selected from a group consisting of a peptide, a functional group,a tag, and a peptide containing a functional group.

Additional Embodiment 4. A compound according to additional embodiment3, wherein Y is either a solubility enhancing tag or an immobilizationtag.

Additional Embodiment 5. A compound according to additional embodiment4, wherein Y is selected from a group consisting of PEG, poly-lysine,poly-arginine, poly-glutamic acid, and poly-aspartic acid.

Additional Embodiment 6. A compound according to additional embodiment4, wherein Y is selected from a group consisting of biotin, hydrazine,aminooxy, azide, alkynyl, alkenyl, aldehyde, pyrroloalanine, carboxy andthiol.

Additional Embodiment 7. A method comprising the steps of

-   a) synthesizing a peptide on a solid support, said peptide    comprising a terminal amino group,-   b) providing a building block according to any one of additional    embodiments 1-2-   c) coupling said building block to said peptide.

Additional Embodiment 8. The method of additional embodiment 7, furthercomprising the steps of

-   d) removing protective group B, and-   e) coupling at least one amino acid building block to the terminal    amino group.

Additional Embodiment 9. The method of additional embodiment 7, furthercomprising the steps of

-   d) removing protective group B,-   e) optionally coupling at least one amino acid building block to the    terminal amino group, and-   f) coupling a tag or a functional group to the terminal amino group.

Additional Embodiment 10. The method of additional embodiment 9, whereinsaid functional group or tag is selected from a group consisting of PEG,poly-lysine, poly-arginine, poly-glutamic acid, poly-aspartic acid,biotin, hydrazine, aminooxy, azide, alkynyl, alkenyl, aldehyde,pyrroloalanine, carboxy and thiol.

Additional Embodiment 11. The method of any one of additionalembodiments 7-10, further comprising the step of

g) removing protective group A at a pH ≤ 6, thereby also removing otherprotective groups present on said peptide and cleaving said peptide fromthe solid support.

Additional Embodiment 12. The method of additional embodiment 11,further comprising the step of

d) cleaving the generated peptide at a pH ≥ 8.

Additional Embodiment 13. The method of any one of additionalembodiments 9-10, wherein said tag or functional group is a solubilizingtag, further comprising the steps of

-   g) removing protective group A at a pH ≤ 6, thereby also removing    other protective groups present on said peptide and cleaving said    peptide from the solid support-   h) purifying said peptide, and i) cleaving off said solubilizing tag    at a pH ≥ 8.

Additional Embodiment 14. The method of any one of additionalembodiments 9-10, wherein said tag or functional group is animmobilizing tag, further comprising the steps of

-   g) removing protective group A at a pH ≤ 6, thereby also removing    other protective groups present on said peptide and cleaving said    peptide from the solid support-   h) immobilizing said peptide via said immobilizing tag on a solid    support-   i) optionally conjugating said peptide to an additional chemical    entity-   j) cleaving off said immobilizing tag at a pH ≥ 8.

Additional Embodiment 15. The method of additional embodiment 14,wherein said chemical entity is a nucleic acid, oligonucleotide ornucleotide, which is preferably a nucleoside-hexaphosphate.

All of the U.S. Patents, U.S. Patent Application Publications, U.S.Patent Applications, foreign patents, foreign patent applications andnon-patent publications referred to in this specification and/or listedin the Application Data Sheet are incorporated herein by reference, intheir entirety. Aspects of the embodiments can be modified, ifnecessary, to employ concepts of the various patents, applications andpublications to provide yet further embodiments.

Although the disclosure herein has been described with reference toparticular embodiments, it is to be understood that these embodimentsare merely illustrative of the principles and applications of thepresent disclosure. It is therefore understood that numerousmodifications may be made to the illustrative embodiments and that otherarrangements may be devised without departing from the spirit and scopeof the present disclosure as defined by the appended claims.

1. A building block comprising the structure:

wherein 2 < n < 24, m = 2 or 3, A is an acid labile protective group,and B is a tag or base labile protective group.
 2. The building block ofclaim 1, wherein A is selected from the group consisting oftert-Butyloxycarbonyl (“Boc”) or Fluorenylmethyloxycarbonyl (“Fmoc”). 3.The building block of claim 1, wherein B is selected from the groupconsisting of Boc or Fmoc.
 4. The building block of claim 1, wherein Ais Boc and B is Fmoc; or A is Fmoc and B is Boc.
 5. The building blockof claim 1, wherein m is
 2. 6. The building block of claim 1, wherein mis
 3. 7. The building block of claim 1, wherein n is
 4. 8. The buildingblock of claim 1, wherein the building block is

.
 9. The building block of claim 1, wherein the building block is

.
 10. A compound comprising the structure:

wherein 2 < n < 24, m = 2 or 3, X is a peptide or a solid support, and Yis selected from the group consisting of a peptide, a functional group,a tag, a peptide including a functional group or a tag, PEG, or a groupincluding PEG.
 11. The compound of claim 10, wherein the tag is selectedfrom the group consisting of a solubility enhancing tag or animmobilization tag.
 12. The compound of claim 10, wherein Y is selectedfrom the group consisting of poly-lysine, poly-arginine, poly-glutamicacid, and poly-aspartic acid.
 13. The compound of claim 10, wherein thefunctional group is selected from the group consisting of hydrazine,aminooxy, azide, alkynyl, alkenyl, aldehyde, pyrroloalanine, carboxy,and thiol.
 14. The compound of claim 10, wherein Y comprises a peptidehaving between 2 and 120 residues.
 15. The compound of claim 10, whereinX and Y both comprise peptides.
 16. The compound of claim 15, wherein mis 2 and n is 2 or
 3. 17. The compound of claim 15, wherein m is 3 and nis 2 or
 3. 18. The compound of claim 10, wherein n is
 2. 19. Thecompound of claim 10, wherein n is
 3. 20. The compound of claim 10,wherein Y comprises a tag and X comprises a peptide.